Grid etcher

ABSTRACT

An apparatus and process for monitoring migratory cell proliferation with restricted migration on a substrate includes providing a substrate, coating the substrate with extracellular matrix, plating cells suspended in cell culture media on extracellular matrix, and placing intersecting channels across the extracellular matrix components by removing the extracellular matrix components from the channels to isolate islands of the extracellular matrix components on the substrate. When the cells are immersed with a fluid, migration of the cells is confined to the isolated islands of the extracellular matrix components, permitting long-term observation of a migratory population.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/967,703, filed on Oct. 14, 2004, and issued as U.S. Pat. No.7,276,367 on Oct. 2, 2007.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

The technology set forth herein is federally sponsored and funded by theUnited States Department of Health and Human Services. The United Statesgovernment has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to monitoring the proliferation of migratorycells such as CNS stem cells/embryonic stem cells or any other migratorycells. More specifically, an apparatus and process is disclosed forplacing isolated islands of an extracellular matrix on a glass coverslip, and, plating, growing, and monitoring the proliferation ofmigratory cells on the isolated islands of the extracellular matrix.

Migratory cells, such as CNS stem cells/embryonic stem cells or anyother migratory cells are grown and simultaneously monitored duringproliferation for research purposes. Typically, an extracellular matrixis placed upon a substrate, usually a coverslip. Thereafter, cells areplated to (placed on) the extracellular matrix for observation duringproliferation. During this observed proliferation, the cellsunfortunately also migrate.

Migration causes problems. Typically, only a discrete area on asubstrate, such as a portion of a substrate having the extracellularmatrix, can be observed. During migration, cells within the observeddiscrete area move out of the observed discrete area; what becomes ofsuch cells is never known to the observer. Other foreign cells,initially outside of the observed discrete area, move into the observedarea; as a consequence, the beginning history of such cells can never beknown to the observer. In either case, the integrity of the monitoringprocesses is degraded. Where one wishes to have an observed history ofthe proliferation of a group of monitored cells, migration of the cellsinto or out of the observed area must be inhibited.

For this reason, where cell populations are grown on substrates, such asglass cover slips, attempts have been made to provide isolated islandsof substrate. These isolated islands of substrate have the effect ofallowing cell migration within their boundaries while maintaining adiscrete cell population within an observable area.

Attempts to create isolated islands has included printing glass coverslips with the desired isolated islands. In this technique, the materialfrom which the extracellular matrix is ultimately formulated is placedupon a printing block having printing areas shaped with the desiredprofile of isolated islands of extracellular matrix. Thereafter, theprinting block is impressed upon a substrate, such as a glass coverslip, to transfer the extracellular matrix material from the print blockto the glass cover slip. Substrate material is transferred from theprinting block onto the glass cover slip much as printer's ink istransferred from a printing block onto paper which is to be printed. Theextracellular matrix transferred to the substrate has the profile of theprinting areas on the printing block.

Other techniques have included photo lithographic masks utilizing photoresist, contact masks, micro stamping, and ink jet printing.

For many of these techniques to work, drying of the substratetransferred on the glass cover slip from the printing block is required.Unfortunately, drying substantially degrades or destroys theextracellular matrix. The growth of cells for subsequent observation isdifficult. Further, many of these techniques leave residual chemicals(such as photo resist) with the deposited substrate. These residualchemicals degrade the subsequent growth on the substrate. Mostimportantly, these techniques do not permit plating of the migratorycells before formation of the islands of substrate.

It has also been contemplated to utilize lasers to form continuouschannels upon a continuous layer of extracellular matrix on a glasscover slip. In this technique, the extracellular matrix is placed upon asubstrate, typically a glass cover slip. Once the extracellular materialis placed, at least one laser is utilized to etch channels in theextracellular matrix. Typically, the extracellular matrix is submergedduring laser etching of channels. This provides isolated islands ofextracellular matrix having separation between isolated islandscomplimentary to the channel width and placement on the substrate.

The technique suffers from several disadvantages.

Utilizing lasers to make such channels is expensive. Further, theablation of the extracellular matrix generates debris and produceslocalized heating at the channel boundaries which can damage theextracellular matrix adjacent the channel boundaries. Further, ifcutting of the channels occurs while the matrix is submerged in aliquid, localized heating of the substrate will occur.

BRIEF SUMMARY OF THE INVENTION

An apparatus and process for monitoring migratory cell proliferationwith reduced migration on a substrate includes providing a substrate,such as a glass coverslip. The substrate is coated with extracellularmatrix. Plating the extracellular matrix with migratory cells thenoccurs. Finally, intersecting channels are placed across the platedextracellular matrix by mechanical etching to form desired isolatedislands for the observation of migratory cell growth. When the platedcells within the isolated islands are immersed with a growth media,migration of the cells is confined to the isolated islands of thesubstrate. By observing the isolated islands during cell growth, cellcharacteristics of migratory cells can be observed.

In the following specification, we use the word “substrate” to describethe surface on which the extracellular matrix is placed. The reader willunderstand that this is almost always a coverslip used for magnifiedobservation of the migratory cell proliferation.

Further, we do not emphasize the normal step of conjugating afluorescent dye to the extracellular matrix in order to render theextracellular matrix visible and provide the necessary light forobserving cell proliferation. As this step is conventional inobservations of cellular proliferation, it will not be further discussedherein.

An advantage of this invention is that the formation of the isolatedislands of extracellular matrix upon a glass cover slip is carried outin a continually moist environment. A continually moist environment canbe assured by immersing the cover slip during the mechanical etching ofthe channels.

In the preferred embodiment, cell plating to the extracellular matrixoccurs before the channels are mechanically etched. There is a reasonfor this order.

If the mechanical etching is done first, and the plating is done second,some cells that settle within the plating area will attach to themechanically etched channels where the extracellular material has beenremoved. The cells within the channel areas, lacking the extracellularmatrix, will become detached and float in the liquid. At least some ofthese cells floating within the liquid will eventually land on surfacesof the substrate that have the extracellular matrix. After such landing,they will attach to the regular growth surface and become normalmigratory cells, indistinguishable from the cell group underobservation. This will introduce new cells into the controlled growthareas which will degrade the controlled observation process of cellproliferation.

By plating the cells on the extracellular matrix, and then removing theplated cells with the extracellular matrix within the channel areas, thephenomena of detached cells floating in the liquid is minimized.

A specialized apparatus is disclosed for mechanically etching regularlyspaced intersecting channels onto the extracellular matrix on thesubstrate. Specifically, an open chamber is defined on the cover slipoverlying the extracellular matrix. This open chamber has a fluid tightboundary about the periphery of the chamber to maintain liquid mediawithin the chamber. An etching tool has an etching end with regularlyspaced flat mechanical etching teeth separated by regularly spacedintervals for etching the extracellular matrix. The chamber is suppliedwith a rectilinear opening having boundaries for guiding the etching endof the etching apparatus in movement across the substrate. The etchingapparatus has a width to fit snugly within the dimension of therectilinear opening.

In use, the chamber is placed over the extracellular matrix on thesubstrate and filled with cell culture media. At least one etching toolwith its regularly spaced flat mechanical etching teeth is fitted to therectilinear opening and extends through the liquid to the substrate. Theetching tool is guided by the rectilinear opening and mechanicallyetches the extracellular matrix while undergoing guided etchingexcursion across the rectilinear opening. By first having a mechanicaletching tool undergo guided etching excursion parallel to first oppositesides of the rectilinear opening and then having a mechanical etchingtool undergo excursion parallel to second opposite sides of therectilinear opening, a regular matrix of channels is defined upon theextracellular matrix. This leaves a pattern of uniformly sized andaligned rectilinear islands upon the extracellular matrix for theisolation of migratory cell growth.

In the normal case, the rectilinear opening is square and a singlemechanical etching tool is utilized. It will be understood that twoetching tools having varying widths and teeth of varying dimension andspacing can be used to generate rectangular growth areas of varieddimensions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view of a holding plate, a cover slip coated withan extracellular matrix placed upon the holding plate, an O-ring, andoverlying cell grid making guide and, an etching tool aligned foretching excursion to configure a first set of channels within theextracellular matrix;

FIGS. 2A and 2B are plan and elevation views of the holding plate,respectively;

FIGS. 3A and 3B are plan views of cell grid making guides;

FIG. 4 is a side elevation of an etching tool for excursion within thecell grid making guide of FIG. 3, the tool having etching members atboth ends;

FIG. 5 is a plan view of multiple restricted-growth areas generated as agrid on an observation cover slip;

FIG. 6 is a plan view of a single rectilinear growth area configured byutilizing a first end of the etching tool shown in FIG. 4 for a verticalchannels and a second end of the etching tool shown in FIG. 4 forhorizontal channels;

FIGS. 7A and 7B are a matrix of unrestrained and restrained growthislands and timed intervals illustrating the cell monitoring permittedby this invention; and,

FIGS. 8A and 8B are a lineage analysis for cells of the unrestrained andrestrained growth islands illustrating the superior monitoring of thisinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the exploded view of FIG. 1, holding plate H is shown withcover slip C overlying the holding plate. Typically, cover slip C has anupwardly exposed surface 10 onto which extracellular matrix 11 isadhered and layered. Typically, this extracellular matrix 11 includestwo layers including poly-L-ornithine which binds to the upper surfaceof glass cover slip C and a covering layer of fibronectin which providesthe surface for attachment of the cells.

The extracellular matrix 11 is a growth surface. In order to effectivelycapture cells and thereafter permit the optimum propagation of thecaptured cells, this surface should preferably remain moist at alltimes. Because of this requirement, it will be seen that the chamber foretching the desired isolated islands is designed to have the O-ringboundary maintaining the surface of cover slip C in a fluid environment.Immediately overlying cover slip C, circular O-ring O provides areservoir barrier immediately underlying grid making guide G.

Grid making guide G fastens at apertures 20 to screws 14 protrudingupwardly of holding plate H. Grid making guide G includes centralbulbous portion having rectilinear aperture A, here shown as a square,configured centrally of the guide. As will hereinafter become apparent,cover slip C is placed on the holding plate H with O-ring O overlyingupwardly exposed surface 10 and extracellular matrix 11. Typically,holding plate H, cover slip C, O-ring O, and grid making guide G are allheld together by wing nuts 16 on screws 14 to provide a gentle but firmcompression on cover slip C toward holding plate H.

Etching tool E includes first etching end 30 and second etching end 31.Each of these etching ends includes seven regularly thick etching tines32 spaced by regular intervals 33. Further, etching tool E has anoverall width which matches the overall width of square aperture Awithin grid making guide G. It will be seen that the tines 32 andintervals 33 vary at each end of etching tool E.

In operation, cover slip C is covered with the extracellular matrix andthereafter plated over the surface of the extracellular matrix with thecells to be analyzed during proliferation. Once this is done, holdingplate H, cover slip C, O-ring O, and grid making guide G are allassembled, and a liquid is poured into the chamber defined between coverslip C and grid making guide G by O-ring O. Thereafter, mechanicaletching occurs utilizing an end 30, 31 of etching tool E. This etchingoccurs by aligning etching tool E parallel to one of the sides ofaperture A and moving the tool normal to its parallel alignment. Thismovement places a group of channels L (see FIG. 5) across the surface ofcover slip C. Thereafter, by rotating etching tool E 90°, and repeatingthe movement of the tool normal to its parallel alignment, a second setof intersecting channels L is placed across cover slip C. Since theseintersecting channels L will be at 90° with respect to the earlieretched channels L, the remaining extracellular matrix will form isolatedislands I or I1(See FIGS. 5 and 6 at respective islands I and I1). Ithas been found that these isolated islands I and I1 permit isolatedpopulations of migratory cells to propagate without substantialmigration either from without the boundaries of the islands or fromwithin the boundaries of the islands to positions exterior of theislands.

Referring to FIG. 2A and 2B, holding plate H includes flat plate 15having respective upstanding bolts 14 mounted thereto. This plate allowsa cover slip C to fit conveniently between upstanding bolts 14.

Referring to FIG. 3A, a first cell grid making guide G is illustrated.Grid making guide G includes central bulbous portion 22 spanned oneither side by wings 21. Apertures 20 accommodating upstanding bolts 14are configured in the ends of the respective wings 21. O-ring O is shownin broken lines with respect to bulbous portion 22 of cell grid makingguide G.

Referring to FIG. 3B, a second cell grid making guide G′ is illustrated.In this embodiment central bulbous portion 22 includes three aperturesA1, A2, and A3.

Referring to FIG. 4, etching tool E is illustrated. Etching tool Eincludes two different etching ends 30, 31. Observing end 30, it can beseen that etching tines 32 across end 30 are all the same dimension.Further, each etching tine 32 is separated from its adjacent etchingtines by regular intervals 33. Comparing end 31 with end 30, it will beseen that the dimensions of the tines 32 and their interval ofseparation 33 are varied. Dimensions are important. By way of example,the respective tines on end 30 are 0.042 in. center to center spacingwith slots 0.022 in there between. Similarly, end 31 includes center tocenter spacing of the tines at 0.038 in. with slots of 0.018 in. therebetween. The etching tool E is approximately 10 mm wide by 100millimeters long and made of 1/16 in stainless steel.

Referring to FIG. 5, the end result of the etching process can be seen.Specifically, isolated islands I of the extracellular matrix areillustrated. Referring to FIG. 6, a rectilinear island I1 isillustrated. Rectilinear islands can be formed by using one end 30 ofetching tool E for etching in one direction, and the other end 31 of theetching tool E for etching in the other direction at 90° to the firstdirection.

Referring to FIGS. 7A and 7B, the remarkably different population resultfrom the growing of migratory cells is illustrated. Specifically, andobserving FIG. 7A, where the surface of the extracellular matrix iswithout islands I, cell distribution is sparse. Conversely, andobserving FIG. 7B, where the surface of the extracellular matrix is withislands I, cell distribution is dense.

During proliferation, the areas in which the cells grow are monitoredcontinuously. Where cells leave the monitored areas, there is no way ofidentifying what happens to the cells. Where the cells arrive into themonitored areas, there is no way of identifying what is the priorhistory of the cells and where they have come from.

Where the islands I set forth in this disclosure are utilized, cells arefor the most part confined to the boundaries of the islands. Presumingthat the entirety of the islands are continuously monitored, departingand arriving cells are essentially eliminated enabling a much morecomprehensive record of proliferation to be maintained.

The results of this latter assurance can be seen in the graphicalrepresentation of FIGS. 8A and 8B. Observing FIG. 8A, it will be seenthat in a lineage diagram, or family tree, of the original cells(approximately six) shown, only two derivatives remaining in the finalframe of the recording. All other cells leave the visual field or die.

Observing FIG. 8B, it will be seen that the restrained populationdiffers radically. The observed population is much more numerous.Furthermore, cells are illustrated can be tracked to the seventhgeneration, as distinguished from a mere three generations.

1. A device for making channels in extracellular matrix on a substratecomprising: a plate to support a substrate coated with extracellularmatrix; a frame placed over said substrate and said plate having anaperture which allows working access to at least a portion of saidextracellular matrix; and, an etching tool having multiple tines which,when said tines traverse across said extracellular matrix in the regiondefined by said aperture, creates channels within said extracellularmatrix.
 2. The device of claim 1 wherein said tines are evenly spaced insaid etching tool and each have a rectangular cross-section.
 3. Thedevice of claim 1 wherein said aperture has a rectangular shape.
 4. Thedevice of claim 1 wherein said frame makes a watertight seal with saidsubstrate which allows said extracellular matrix to be maintained in aliquid media.
 5. The device of claim 1 wherein said etching tool has twosets of tines which can be used for removing extracellular matrix.
 6. Anetching assembly comprising: a plate capable of supporting a substrate;a frame having an aperture which is capable of attaching over asubstrate to said plate; and, an etching tool having multiple tineswherein said tines can penetrate through said frame aperture to becapable of etching material away from an underlying substrate.
 7. Theetching assembly of claim 6 wherein said etching tool tines are evenlyspaced and each have a rectangular cross section.
 8. The etchingassembly of claim 6 wherein said aperture has a rectangular shape andsaid frame is attached to said plate with screws.
 9. The etchingassembly of claim 6 further comprising a substrate coated withextracellular matrix.
 10. The etching assembly of claim 6 wherein awatertight seal is formed in said frame aperture when said frame isaffixed over a substrate to said plate.
 11. A process for makingchannels in extracellular matrix on a substrate comprising the steps of:providing a planar substrate; coating said substrate with extracellularmatrix; selecting an etching device comprising a frame with an apertureand an etching tool with multiple tines; projecting the tines of saidetching tool through said frame aperture and using said tines to removechannels of extracellular matrix on said substrate within the regiondefined by said frame aperture.
 12. The process of claim 11 wherein saidtines are evenly spaced and have a rectangular cross-section.
 13. Theprocess of claim 11 wherein channels of extracellular matrix are removedin an intersecting pattern to create islands of extracellular matrix.14. A process for making channels in extracellular matrix with cells ona substrate comprising the steps of: providing a planar substrate;coating said substrate with extracellular matrix; plating migratorycells onto the extracellular matrix to create a matrix with cells;allowing the cells to adhere to the extracellular matrix; selecting anetching device comprising a frame with an aperture and an etching toolwith multiple tines; projecting the tines of said etching tool throughsaid frame aperture and using said tines to remove channels ofextracellular matrix on said substrate within the region defined by saidframe aperture.
 15. The process of claim 14 wherein said tines areevenly spaced and have a rectangular cross-section.
 16. The process ofclaim 14 wherein channels of extracellular matrix are removed in anintersecting pattern to create islands of extracellular matrix.